The present invention relates to new functional ganglioside derivatives and more precisely to new esters and amides, their preparation procedures, pharmaceutical preparations containing the ganglioside esters and amides and to the therapeutic use of the ganglioside esters and amides.
Gangliosides are natural products contained in various animal tissues or organs, above all in the tissues of the central and peripheral nervous systems, but also in the adrenal marrow, in the erythrocytes, in the spleen and elsewhere, from which they can be extracted in a purified form. It has been possible to establish the basic structure of most gangliosides thus obtained. They are glycosphingolipids, that is, compounds resulting from the union of an oligosaccharide with a sphingosine and a certain number of sialic acids bound together by glucosidic or ketosidic bonds. The gangliosides so far described in the literature and obtained in purified form do not represent unitary chemical compounds, except possibly for their saccharide part (oligosaccharide), as the ceramide and sialic components are quite variable, within certain limits. Thus, even when "pure" gangliosides are referred to, this expression is to be broadly interpreted to mean a ganglioside species in which at least a part, for instance the saccharide part, is unitary and characteristic from a chemical point of view. Given this, before describing in more detail the background of the present invention, it is useful to take note of the following general formula which includes all the structures of the gangliosides so far obtained in purified form, and emphasizes the functions which are functionally modified according to the invention (formula I). ##STR1## In this formula, an oligosaccharide residue, formed by a maximum of 5 monosaccharides, is connected by a glucoside bond to a ceramide residue and to one or more sialic acid residues, both by means of as many direct glucoside bonds, and by means of one or more such bonds, as the remaining sialic acid residues are joined together by ketoside bonds. The formula shows the hydroxyl groups of the saccharide portion, of the sialic acids and of the ceramide, as well as the above mentioned glucoside bonds with sialic acids and ceramide and the carboxyl groups of the sialic acids. The sialic acids which form part of the gangliosides of formula I have the general structure II ##STR2## in which one or more of the primary and secondary hydroxyl groups may also be acylated and in which the acyl groups derive from acetic or glycolic acid. The number of sialic acids present in gangliosides usually varies from 1 to 5.
The sialic residues are bound to the oligosaccharide by a ketosidic bond formed by the hydroxyl in position 2 with an oligosaccharide hydroxyl. When several sialic acids are bound together, their molecules are united by means of ketoside bonds formed among the hydroxyls of positions 2 and 8 of two sialic acid molecules. The sialic acids of gangliosides, including purified gangliosides as described above, are mixtures of various chemically unitary acids, such as N-acetylneuraminic and N-glycolylneuraminic acids, in which the first is predominant, and possibly of one or more of their O-acylderivatives, such 8-0-acylderivatives.
Gangliosides are to be found in nature as metallic salts, such as sodium salts, and it is therefore the carboxylic function/s of sialic acids which are salified. The free forms of gangliosides may be easily obtained by treatment of the salts, such as sodium salts, which an acid type ionic exchanger, using for example a resin such as Dowex AG 50.times.8, H.sup.+ form.
The ceramide residue in the gangliosides of formula I generally represents several N-acyl sphingosine having one of the formulas ##STR3## in which n=10-16 and the acyl derives from a saturated or unsaturated fatty acid having between 16 and 22 carbon atoms, or from a corresponding hydroxyacid.
The oligosaccharide is made up of a maximum of 5 monosaccharides or their derivatives with an acylaminic group, especially of hexoses and their derivatives of the above mentioned type. At least one glucose or galactose molecule is, however, always present in the oligosaccharide. The most frequent residue present as an acylaminic derivative of the above mentioned sugars is N-acetylgalactosamine or N-acetylglucosamine.
In order to better illustrate the structure of the gangliosides included in formula I and in particular the character of the bonds between the saccharide parts, the sialic acids and ceramide, reproduced below is the formula of a "pure" GM.sub.1 ganglioside containing only one sialic acid (represented by N-acetylaminic or N-glycolylneuraminic acid). ##STR4##
It is well known that gangliosides are functionally important in the nervous system and it has recently been shown that gangliosides are useful in the therapy of peripheral nervous system pathologies. The therapeutic action of gangliosides seems to consist above all in stimulating sprouting phenomena of the nervous cell and in activating the membrane enzymes involved in the conduction of nervous stimuli, such as the enzyme (Na.sup.+, K.sup.+) ATPase. Neuronal sprouting stimulated by gangliosides promotes functional recovery of the damaged nervous tissue.
Further studies have been carried out to find compounds which could prove more efficient than gangliosides in the therapy of nervous system pathologies. These studies have led for example to the discovery that internal esters of gangliosides, in which one or more hydroxyls of the saccharide part are esterified with one or more carboxylic groups of the sialic acids (intramolecular reaction) with the formation of as many lactonic rings, are more active than gangliosides themselves in promoting neuronal sprouting and in activating membrane enzymes involved in the conduction of nervous stimuli, such as the enyzme NA.sup.+, K.sup.+) ATPase (see U.S. Pat. No. 4,476,119).
According to the present invention, another group of ganglioside derivatives has now been discovered, which presents advantages over gangliosides themselves, inasmuch as they have a prolonged activity in time ("retard" effect). These compounds are derivatives in which the carboxyl group of the sialic acids are functionally modified by esterification or by conversion into amides and derivatives of those esters or amides in which the hydroxyl groups of the saccharide part, of the sialic acids and of the ceramide are also esterified with organic acids, or rather acylate derivatives (which shall be simply called "acylates" hereinafter, and specifically "acetylates, propionylates, etc."). The derivatives of the invention also include ganglioside derivatives in which only the hydroxyl groups are esterified with organic acids, that is, contain free carboxyl groups.
Two methyl esters of the carboxyl of a sialic acid of gangliosides were described in the article "Notes on improved procedures for the chemical modification and degradation of glycosphyngolipids" in the Journal of Lipid Research 21, 642-645 (1980) by MacDonald et al. These compounds are the methyl esters of the gangliosides G.sub.M1 and G.sub.M3 [(the abbreviations used herein to identify gangliosides are those proposed by Svennerholm in J. Jeurochem. 10, 613 (1963))]. However, MacDonald et al. do not report any biological activity for the compounds. The methyl ester of the ganglioside G.sub.M3 is also used in the preparation of one of its peracylate derivatives for use in several degradation or copulation reactions [Methods of Enzymology, 50, 137-140 (1978)]. The above mentioned article in Journal of Lipid Research by MacDonald et al. also describes an acetylation of the methyl esters of gangliosides G.sub.M1 and G.sub.M3, but without isolating the acylated compounds.
Acetylation with acetic anhydride-pyridine of lipids extracted from the spleen, liver and kidneys of Buffalo rats, from Morris hepatomas and from fibroblast cells was described by Terunobo Saito and Sen-Itiroh Hakomori in the Journal of Lipid Research, 12, 257-259, (1971). The authors isolated by chromatography the acetylated mixture of the gangliosides contained in those lipids from the acylated product, together with other glycolipids without, however, identifying any specific ganglioside. Of the amides, the unsubstituted amide of the ganglioside G.sub.M3 was described [Ad. Exp. Med. Biol. 19, 95 (1972)], but even in this case, no biological properties were mentioned.